Method and system for determining the concentration of an analyte in a fluid sample

ABSTRACT

A method and system are provided for detecting the concentration of an analyte in a fluid sample. The method and system involve analysis of a volatilized, ionized fluid sample using a mass spectrometer or other ionic analyte detection device that provides a signal proportional in intensity to the quantity of ionized analyte detected. The improvement involves replacement of a necessary non-analyte component in the fluid sample with a substitute component that serves the same purpose as the original component but is either more volatile than the original component and/or the analyte or undergoes a reaction to provide lower molecular weight reaction products, and results in an increased intensity in signal and signal-to-noise ratio. Acoustic fluid ejection is a preferred method of generating nanoliter-sized droplets of fluid sample that are then volatilized, ionized, and analyzed. Also provided are zwitterionic compounds suitable as the substitute components that when ionized and heated decompose to provide carbonic dioxide, a nitrogenous species such as ammonia, an amine, or nitrogen gas, and a volatile aromatic compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 14/887,320, filed Oct.19, 2015, which claims priority under 35 U.S.C. § 119(e)(1) toprovisional U.S. Patent Application Ser. No. 62/065,600, filed Oct. 17,2014, the disclosures of which are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates generally to improved methods fordetecting the presence or quantity of an analyte in a fluid sample, andmore particularly relates to improvements in such methods wherenanoliter-sized fluid droplets are generated and the sample accuratelyanalyzed. The invention finds utility in the fields of analyticalchemistry, biological research, pharmaceuticals, and medicine.

BACKGROUND

Accurate determination of the presence, identity, concentration, and/orquantity of an ionized species in a sample is critically important inmany fields. Most techniques used in such analyses involve ionization ofspecies in a fluid sample prior to introduction into the analyticalequipment employed. The choice of ionization method will depend on thenature of the sample and the analytical technique used, and manyionization methods are available, including, without limitation,chemical ionization, electron impact ionization, desorption chemicalionization, and atmospheric pressure ionization, including electrosprayionization and atmospheric pressure chemical ionization.

The presence of contaminants in biological samples undergoing analysisis obviously problematic for a number of reasons. Contaminants mightcause interference with the analytical procedure, chemically orphysically altering the sample or the analyte itself. Contaminants mayalso be mistaken for analyte or vice versa, such that the concentrationmeasured concentration of analyte may be significantly higher or lowerthan the actual concentration. The same problem can be caused bynecessary components of the fluid sample, such as buffer salts,surfactants, and other species that may be essential for biochemicalsteps preceding the analysis.

Mass spectrometry is a well-established technique that involves thedetection of an analyte in ionized form. In this technique, samplemolecules are ionized and the resulting ions are sorted bymass-to-charge ratio. For analytes contained in a fluid sample, thesample is typically converted to an aerosol that undergoes desolvation,vaporization, and ionization in order to form fluid ions.

The presence of non-analyte species can be particularly problematic inmass spectrometry, where analyte concentration may be very low and thenumber and concentration of contaminants and interfering components maybe relatively high. One study has documented over 650 contaminants andinterfering components frequently found in biological samples undergoingmass spectrometric analysis. B. O. Keller et al. (2008), “Interferencesand contaminants encountered in modern mass spectrometry,” Anal. Chim.Acta 627(1):71-81. These include salts, buffering agents, endogenouscompounds, surfactants, drugs, metabolites, and proteins. When thesespecies reduce detection sensitivity by decreasing the signal-to-noiseratio and give rise to a flawed analysis, the problem has beencharacterized as “ion suppression.” See Weaver et al. (2006) RapidCommunications in Mass Spectrometry 20:2559-64.

It has been postulated that “the main cause of ion suppression is achange in the spray droplet solution properties caused by the presenceof nonvolatile or less volatile solutes,” i.e., solutes that arenonvolatile or less volatile than the analyte; see Annesley (2003) “IonSuppression in Mass Spectrometry,” in Clinical Chemistry49(7):1041-1044, citing King et al. (2000) J. Am. Soc. Mass Spectrom.11:942-50. The reference explains that the nonvolatile or less volatilecontaminants and components change the efficiency of droplet formationor droplet volatilization, which in turn affects the quantity of chargedanalyte in the gas phase that ultimately reaches the detector. Annesleycites studies showing that molecules of higher mass tend to suppress thesignal of smaller molecules and that more polar analytes are susceptibleto ion suppression. Annesley at 1042, citing Sterner et al. (2000) J.Mass Spectrom. 35:385-91 and Bonfiglio et al. (1999) Rapid Commun. MassSpectrom. 13:1175-85. Weaver et al. cites several possible mechanismsunderlying ion suppression: (1) competition for charge between analyteand ion-suppressing agent, leading to reduced ionization of analyte; (2)large concentrations of ion-suppressing agents causing an increase insurface tension as well as an increase in droplet viscosity, in turnresulting in decreased evaporation efficiency; and (3) gas phasereactions between the ionized analyte and other species in the sample,resulting in an overall loss of charge from the analyte ions. Weaver etal. at 2562.

As electrospray ionization (ESI) has a relatively complex ionizationmechanism, relying heavily on droplet charge excess, there areadditional factors to consider when exploring the cause of ionsuppression and potential solutions. It has been widely observed thatfor many analytes, at high concentrations, ESI exhibits a loss ofdetector response linearity, perhaps due to reduced charge excess causedby analyte saturation at the droplet surface, inhibiting subsequentejection of gas phase ions from further inside the droplet. Thus,competition for space and/or charge may be considered as a source of ionsuppression in ESI. Both physical and chemical properties of analytes(e.g. basicity and surface activity) determine their inherent ionizationefficiency. Biological sample matrices naturally tend to contain manyendogenous species with high basicity and surface activity, and thetotal concentration of these species in the sample will thus quicklyreach levels at which ion suppression can be expected.

Another explanation of ion suppression in ESI considers the physicalproperties of the droplet itself rather than the species present. Asnoted above, high concentrations of interfering components give rise toincreased surface tension and viscosity that in turn reduce evaporationefficiency, and this is known to have a marked effect on ionizationefficiency.

An additional theory to explain ion suppression in ESI relates to thepresence of non-volatile species that can either cause co-precipitationof analyte in the droplet (thus preventing ionization) or prevent thecontraction of droplet size to the critical radius required for ionevaporation and/or charge residue mechanisms to form gas phase ionsefficiently. It should also be pointed out that the degree of ionsuppression may be dependent on the concentration of the analyte beingmonitored, and with the ever-increasing demand to lower detectionthreshold, ion suppression may become a more and more serious problem.

Ion suppression has primarily been addressed by de-salting the fluidsample using dialysis, liquid chromatography, solid-phase extraction, orion exchange. These processes require time, materials, and equipment,and can reduce the available quantity of an already small sample. Inaddition, certain ionic or ionizable species may be essential tomaintain in the sample, such as buffer systems.

An ideal method to address ion suppression would:

Eliminate the need for additional process steps and materials, includingclean-up and de-salting;

Eliminate the need for additional processing time;

Be adaptable to use with very small sample sizes, consume a smallportion of the small sample size, allow for detection of low analyteconcentrations, and be composed of very small droplets; and

Be capable of implementation in a high speed analytical system such ashigh throughput mass spectrometry, optimally enabling analysis of up toat least 50,000 samples per day or more; and

Eliminate the need for pre-analysis “clean-up” of the sample to removecontaminants and interfering components.

SUMMARY OF THE INVENTION

Accordingly, the present invention addresses the aforementioned need inthe art by providing an improved method for accurately determining theconcentration of an analyte in a fluid sample.

In one embodiment, an improved method is provided for determining theconcentration of an analyte in a fluid sample that also contains anecessary non-analyte component, where the method comprises volatilizingand ionizing the fluid sample, and introducing the ionized, volatilizedsample into an ionic analyte detection device that provides an analytesignal proportional in intensity to the quantity of analyte detected,e.g., a mass spectrometer, wherein the improvement comprises replacingthe necessary non-analyte original component with a substitute componentthat (a) functions as the necessary non-analyte component in the fluidsample, i.e., serves the same purpose with respect to the fluid sample,(b) is more volatile than the original component and/or the analyte,and/or undergoes a chemical reaction to yield at least one reactionproduct that is more volatile than the original component and/or theanalyte, and (c) results in an increase in the intensity of the analytesignal and/or a greater signal to noise ratio than either the signalintensity or signal to noise ratio obtained using the originalcomponent.

In another embodiment, acoustic ejection is used to generatenanoliter-sized droplets that are then volatilized, ionized, andanalyzed, wherein “nanoliter-sized” droplets are defined herein asdroplets of 5 nl or less. In acoustic ejection, an acoustic ejectordirects focused acoustic energy into a reservoir containing the fluidsample in a manner that results in the ejection of fluid droplets fromthe surface of the fluid sample. Acoustic ejection provides manyadvantages over other droplet generation methodologies; for instance,acoustic fluid ejection devices are not subject to clogging, misdirectedfluid or improperly sized droplets, and acoustic technology does notrequire the use of tubing or any invasive mechanical action. Acousticejection technology as described, for example, in U.S. Pat. No.6,802,593 to Ellson et al., enables rapid sample processing andgeneration of droplets in the nanoliter or even picoliter range. Inaddition, acoustic ejection enables control over droplet size as well asrepeated generation of consistently sized droplets. See U.S. Pat. No.6,416,164 B1 to Stearns et al., incorporated by reference herein. Asexplained in that patent, the size of acoustically ejected droplets froma fluid surface can be carefully controlled by varying the acousticpower, the acoustic frequency, the toneburst duration, and/or theF-number of the focusing lens.

In a further embodiment, a zwitterionic compound is used as thesubstitute non-analyte component in the method of the invention. Uponvolatilization, the zwitterionic compound undergoes a chemical reactionto yield at least one reaction product that is more volatile than theoriginal non-analyte component and/or the analyte.

In another embodiment, a method is provided an improved method isprovided for determining the concentration of an analyte in a fluidsample that also contains a necessary non-analyte component, where themethod comprises volatilizing and ionizing the fluid sample, andintroducing the ionized, volatilized sample into an ionic analytedetection device that provides an analyte signal proportional inintensity to the quantity of analyte detected, where a substitutecomponent is selected to replace the necessary non-analyte componentand, upon volatilizing the fluid sample, undergoes a reaction to yieldat least one reaction product that is more volatile than the necessaryoriginal component and/or the analyte.

In another embodiment, a method is provided as above wherein theaforementioned reaction is a decomposition reaction.

In a further embodiment, a method is provided as above wherein thereaction involves chemical, photolytic, or thermal cleavage of a linkagein the substitute component that provides lower molecular weightreaction products that do not cause any significant ion suppressionand/or are more volatile than the original component.

In still a further embodiment, a system is provided for determining theconcentration of an analyte in a fluid sample that comprises a massspectrometer, an acoustic ejector to generate droplets of fluid sample,and a means for volatilizing and ionizing the droplets prior tointroduction into the mass spectrometer, the improvement which comprisesreplacing at least one necessary component in the fluid sample with asubstitute component that serves the same function as the originalcomponent but results in an increase in intensity of analyte signaland/or an increase in signal-to-noise ratio relative to the intensity ofthe analyte signal and/or signal-to-noise ratio, respectively, obtainedusing the original component.

In another embodiment, the substitute component of the system contains alinkage that can be chemically, thermally, or photolytically cleaved toprovide lower molecular weight reaction products that do not cause anysignificant ion suppression and/or are more volatile than the originalcomponent. When the substitute component contains a photolyticallycleavable linkage, the system further includes a source of radiationeffective to cleave the linkage.

The method and system of the invention generally provide for an increasein the intensity of analyte signal and/or a greater signal-to-noiseratio that is at least 10% and preferably at least 25% relative to theintensity of the analyte signal and the signal-to-noise ratio obtainedwithout the substitute component.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of normalized signal at m/z=195 versus ammonium acetate(♦) or sodium chloride (▪) concentration (mM), as described inconnection with the mass spectrometric detection of caffeine describedin Example 1.

FIG. 2 is a plot of signal intensity for detected caffeine analyteversus salt concentration for six different buffer salts, as describedin Example 2.

FIG. 3 is a plot of luminescence versus time for the kinase assaycarried out in Example 3, conducted using a volatile magnesium salt(magnesium acetate) and, for purposes of comparison, a nonvolatilemagnesium salt (magnesium chloride).

FIG. 4 schematically illustrates the synthesis of cleavable buffercompound (30).

FIG. 5 schematically illustrates the synthesis of cleavable buffercompound (31), using two alternate routes.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Specific terminology of particularimportance to the description of the present invention is defined below.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, “an analyte” refers not only to asingle analyte but also to a combination of two or more differentanalytes, “a substitute component” refers to a single such component orto a plurality (e.g., a mixture) of components, and the like.

The term “ionizable” as used herein refers to a species that is capableof undergoing ionization. As such, an “ionizable” species herein may bein electronically neutral form or in ionized (or “ionic”) form, as anindividual ion or as a component of a salt. The ionizable species mayalso be present in both electrically neutral and ionized forms, such aswill be the case in a buffer system. As an example, acetic acid(CH₃COOH) is an ionizable compound that may be present in electronicallyneutral form, with a protonated carboxyl group, or it may be present inionized form, with the proton removed to give the acetate ion (CH₃COO⁻),or it may be present as a combination of the electronically neutral andionized forms. As another example, a zwitterion containing a carboxylicacid group and an amino group may be in electrically neutral form or inionized form in which the carboxylic acid group is ionized tocarboxylate and the amino group is protonated to give a cationicnitrogen-containing substituent.

The term “volatile” is used herein to refer to the relative tendency ofan ion, salt, or compound to leave the surface of a fluid droplet andenter the vapor phase under the vaporization conditions and using thevaporization methods discussed herein. The term is used in a comparativesense herein, such that the substitute species is “volatile” insofar asit is more likely than either the original non-analyte component or theanalyte to volatilize under the volatilization conditions employed inconjunction with described method.

The terms “contaminant” and “component” are used to refer to species inthe fluid sample that cause ion suppression. The term “contaminant,”however, refers to a species that is unintentionally or accidentallyintroduced into the sample and may have been present in a solvent,reagent, surfactant, or the like, while the term “component” refers to aspecies that serves a necessary and intended purpose, such as speciesthat are required for biochemical processing preceding the analysisand/or species that are necessary to maintain a chemical or physicalparameter of the fluid sample, e.g., buffer systems to maintain pH.

The terms “acoustic radiation” and “acoustic energy” are usedinterchangeably herein and refer to the emission and propagation ofenergy in the form of sound waves. As with other waveforms, acousticradiation may be focused using a focusing means, as discussed below.

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point, either bya device separate from the acoustic energy source that acts like a lens,or by the spatial arrangement of acoustic energy sources to effectconvergence of acoustic energy at a focal point by constructive anddestructive interference. A focusing means may be as simple as a solidmember having a curved surface, or it may include complex structuressuch as those found in Fresnel lenses, which employ diffraction in orderto direct acoustic radiation. Suitable focusing means also includephased array methods as are known in the art and described, for example,in U.S. Pat. No. 5,798,779 to Nakayasu et al. and Amemiya et al. (1997)Proceedings of the 1997 IS&T NIP 13 International Conference on DigitalPrinting Technologies, pp. 698-702.

The terms “acoustic coupling” and “acoustically coupled” used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two items are indirectly acoustically coupled, an “acousticcoupling medium” is needed to provide an intermediary through whichacoustic radiation may be transmitted. Thus, an ejector may beacoustically coupled to a fluid, e.g., by immersing the ejector in thefluid or by interposing an acoustic coupling medium between the ejectorand the fluid to transfer acoustic radiation generated by the ejectorthrough the acoustic coupling medium and into the fluid.

The term “reservoir” as used herein refers to a receptacle or chamberfor holding or containing a fluid. Thus, a fluid in a reservoirnecessarily has a free surface, i.e., a surface that allows a droplet tobe ejected therefrom. In its one of its simplest forms, a reservoirconsists of a solid surface having sufficient wetting properties to holda fluid merely due to contact between the fluid and the surface.

The term “fluid” as used herein refers to matter that is nonsolid or atleast partially gaseous and/or liquid. A fluid may contain a solid thatis minimally, partially or fully solvated, dispersed or suspended.Examples of fluids include, without limitation, aqueous liquids(including water per se and salt water) and nonaqueous liquids such asorganic solvents and the like.

The invention accordingly provides an improved method for determiningthe concentration of an analyte in a fluid sample, where the fluidsample contains, in addition to the analyte, a necessary non-analytecomponent, for instance a buffer system including a buffer salt formaintaining pH or a salt for maintaining ionic strength, and wherein themethod involves volatilizing and ionizing the fluid sample andintroducing the ionized, volatilized sample into an ionic analytedetection device, such as a mass spectrometer, that generates an analytesignal proportional in intensity to the quantity of ionized analytedetected. The improvement provided by the invention involves employing asubstitute component for the original non-analyte component that willserve the same purpose as the original component but is more volatilethan the original component and/or the analyte or undergoes a chemicalreaction upon volatilization to yield a reaction product that is morevolatile than the original component and/or the analyte. The substitutecomponent results in a stronger analyte signal and/or an increasedsignal-to-noise ratio relative to the analyte signal and signal-to-noiseratio obtained with the original non-analyte component. Preferredsubstitute components, at least in part because of volatilityconsiderations, increase the signal-to-noise ratio by at least 20%, andparticularly preferred such components increase the signal-to-noiseratio by 50% or more. The purpose of the necessary non-analyte componentmay be, as noted, maintaining a pre-determined pH or a required ionicstrength.

The analyte in the fluid sample may be any analyte of interest. Examplesof analytes include, without limitation, drugs, metabolites, inhibitors,ligands, receptors, catalysts, synthetic polymers, and allostericeffectors. Often, the analyte is a “biomolecule,” i.e., any organicmolecule, whether naturally occurring, recombinantly produced,chemically synthesized in whole or in part, or chemically orbiologically modified, that is, was or can be a part of a livingorganism. The term encompasses, for example, nucleotidic analytes,peptidic analytes, and saccharidic analytes.

Nucleotidic analytes may be nucleosides or nucleotides per se, but mayalso comprise nucleosides and nucleotides containing not only theconventional purine and pyrimidine bases, i.e., adenine (A), thymine(T), cytosine (C), guanine (G) and uracil (U), but also protected formsthereof, e.g., wherein the base is protected with a protecting groupsuch as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl,and purine and pyrimidine analogs. Suitable analogs will be known tothose skilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,1-methyladenine, 2-methyladenine, N⁶-methyladenine,N⁶-isopentyl-adenine, 2-methylthio-N.sup.6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromo-guanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluoro-uracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methyl-aminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine. In addition, the terms “nucleoside” and “nucleotide”include those moieties that contain not only conventional ribose anddeoxyribose sugars, but other sugars as well. Modified nucleosides ornucleotides also include modifications on the sugar moiety, e.g.,wherein one or more of the hydroxyl groups are replaced with halogenatoms or aliphatic groups, or are functionalized as ethers, amines, orthe like.

Nucleotidic analytes also include oligonucleotides, wherein the term“oligonucleotide,” for purposes of the present invention, is generic topolydeoxyribo-nucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones. Thus, anoligonucleotide analyte herein may include oligonucleotidemodifications, for example, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.). There is no intended distinction in length between theterms “polynucleotide” and “oligonucleotide,” and these terms are usedinterchangeably. These terms refer only to the primary structure of themolecule. As used herein the symbols for nucleotides and polynucleotidesare according to the IUPAC-IUB Commission of Biochemical Nomenclaturerecommendations (Biochemistry 9:4022, 1970).

“Peptidic” analytes are intended to include any structure comprised ofone or more amino acids, and thus include peptides, dipeptides,oligopeptides, polypeptides, and proteins. The amino acids forming allor a part of a peptidic analyte may be any of the twenty conventional,naturally occurring amino acids, i.e., alanine (A), cysteine (C),aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G),histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M),asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W), and tyrosine (Y), as well asnon-conventional amino acids such as isomers and modifications of theconventional amino acids, e.g., D-amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically modified aminoacids, β-amino acids, constructs or structures designed to mimic aminoacids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, lacticacid, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline,0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, and nor-leucine), and other non-conventional aminoacids, as described, for example, in U.S. Pat. No. 5,679,782 toRosenberg et al. Peptidic analytes may also contain nonpeptidic backbonelinkages, wherein the naturally occurring amide —CONH— linkage isreplaced at one or more sites within the peptide backbone with anon-conventional linkage such as N-substituted amide, ester, thioamide,retropeptide (—NHCO—), retrothioamide (—NHCS—), sulfonamido (—SO₂NH—),and/or peptoid (N-substituted glycine) linkages. Accordingly, peptideanalytes can include pseudopeptides and peptidomimetics. Peptideanalytes can be (a) naturally occurring, (b) produced by chemicalsynthesis, (c) produced by recombinant DNA technology, (d) produced bybiochemical or enzymatic fragmentation of larger molecules, (e) producedby methods resulting from a combination of methods (a) through (d)listed above, or (f) produced by any other means for producing peptides.

Saccharidic analytes include, without limitation, monosaccharides,disaccharides, oligosaccharides, polysaccharides, mucopolysaccharides orpeptidoglycans (peptido-polysaccharides) and the like.

An exemplary embodiment of the invention involves replacement of astandard, relatively nonvolatile buffer salt associated with ionsuppression by a substitute buffer salt that is more volatile than thestandard buffer salt and provides for reduced ion suppression asevidenced by an increased analyte signal and/or an increasedsignal-to-noise ratio. It is established that relatively nonvolatilesalts in fluid samples are frequent contaminants that can cause ionsuppression and thus imprecise or incorrect results.

In this embodiment, the necessary non-analyte component is the saltcomponent of a standard buffer system, and the substitute component is amore volatile salt that serves the same purpose, i.e., maintains thesame pH, and that reduces or eliminates the ion suppression seen withthe standard buffer salt. In general, salts of strong acids or bases arenot sufficiently volatile for the present purpose. For instance, sodiumcations, potassium cations, calcium cations, and tetrabutylammoniumcations should be avoided, as should anions such as sulfates andnitrates. A notable exception is hydrochloric acid, which although astrong acid in water, is a relatively weak acid in the gas phase.Further, ions bearing multiple charges, such as sulfates, citrates andphosphates, are not likely to be volatile. Finally, higher molecularweight salts such as fatty acid salts are not likely to be volatileexcept at very high temperatures.

Examples of relatively volatile buffer salts that may replace theconventional buffer salts, or original non-analyte component, include,without limitation, the following:

Ammonium bicarbonate

Ammonium formate

Ammonium acetate

Ammonium propionate

Ammonium butyrate

Pyridinium formate

Pyridinium acetate

Pyridinium propionate

Pyridinium butyrate

Ethylmorpholinium acetate

Dimethylamino formate

Dimethylamino acetate

Dimethylamino propionate

Dimethylamino butyrate

Methylethylamino formate

Methylethylamino acetate

Methylethylamino propionate

Methylethylamino butyrate

Diethylamino formate

Diethylamino acetate

Diethylamino propionate

Diethylamino butyrate

Within the aforementioned group, preferred volatile buffer salts includethe following:

Ammonium bicarbonate

Ammonium formate

Ammonium acetate

Pyridinium acetate

Pyridinium formate

Ethylmorpholinium acetate

Trimethylamino acetate

Trimethylamino formate

It is to be understood that the aforementioned salts are merelyrepresentative, and that other salts may also be used, providing thatthey serve the same purpose as the salt they are replacing and that theymeet the volatility and enhanced signal-to-noise criteria set forthherein.

Candidate salts may be readily tested for volatility using methods wellknown to those of ordinary skill in the art. Such methods include, forexample, a dry residue analysis, in which the candidate salt or buffercomposition is placed in a volatile solvent and then heated to dryness.The presence of any dry residue suggests that the salt is notsufficiently volatile for use in the present purpose. Those candidatesalts established as sufficiently volatile are then tested for theircapability to reduce ion suppression, by conducting a comparison of thecandidate salt with the necessary non-analyte component the candidatesalt is intended to replace. Volatility may also be evaluated usingbutyl acetate number, a measure of relative evaporation rates, as willbe appreciated by those in the field.

Generally, although not necessarily, the ionic detection device is amass spectrometer. It will be appreciated that various volatilizationtechniques are available in connection with mass spectrometry, includingthermal methods and electrospray, and any effective volatilizationtechnique may be used in conjunction with the present method. Any of anumber of known ionization means may also be used, including chemicalionization, field desorption ionization, electrospray ionization,atmospheric pressure chemical ionization, matrix-assisted laserdesorption ionization, and inductively coupled plasma ionization, and,again, any effective ionization technique may be advantageously employedherein. Depending on the nature of the analyte, mass spectrometricmeasurements can be performed in negative or positive mode, with acidicanalytes preferentially ionizing in the negative mode and basic analytespreferentially ionizing in the positive mode.

In a preferred embodiment, the improved method of the invention employsacoustic ejection to produce very small droplets that are thenvolatilized, ionized, and analyzed. These small droplets arenanoliter-sized droplets, defined herein as a droplets containing atmost about 5 nl of fluid sample, preferably not more than about 2.5 nl,more preferably less than 1 nl, most preferably smaller than about 50pl, and optimally less than about 1 pl. Acoustic ejection of dropletsfrom the surface of a fluid sample is effected using an acoustic ejectoras will be described in detail below. Acoustic ejection technology isparticularly suited to high-throughput mass spectrometry (HTMS), insofaras HTMS has been hampered by the lack of easily automated samplepreparation and loading, the need to conserve sample, the need toeliminate cross contamination, the inability to go directly from a fluidreservoir into the analytical device, and the inability to generatedroplets of the appropriate size.

The present method has proved to be unexpectedly effective withnanoliter-sized droplets, as seen in the Examples herein. It is wellunderstood that fundamental fluid physics changes as the size scale isdecreased, i.e., as fluid droplets become smaller and smaller. Otherwiseapplicable principles of diffusion and mixing tend not to apply tonanoliter-sized droplets, nor are conventional analyses workable whenattempting to predict the flow dynamics of an element, ion, or compoundmoving from the interior of a nanoliter-sized droplet to the dropletsurface, e.g., for purposes of volatilization.

Acoustic ejection of droplets that are then volatilized providesnumerous advantages. In volatilizing droplets that are nanoliter-sized,e.g., using a thermal volatilization technique, the large surface areaof these small droplets facilitates the gas phase extraction of therelatively volatile substitute component, leaving behind the chargedanalyte in the evaporating droplet. Thus, by enabling gas phaseextraction of the substitute component, e.g., the substitute buffer saltor the like, acoustic ejection eliminates the need for liquid phase orsolid phase “clean up” of a fluid sample to remove interferingcomponents that cause ion suppression. This significantly increases thenumber of samples that can be analyzed using mass spectrometry or thelike in a given period of time. With current commercially availablemethodology, the need for an additional step to remove buffer, othersalts, detergents, and any other non-analyte species results in aprocessing time in the range of 7-20 seconds per sample, while thepresent invention enables processing time of well under 1 second persample, typically on the order of about 0.3 seconds per sample.Combining this feature with the fact that the present process can becarried out using far smaller sample sizes and with the fact that theanalyte signal obtained is increased by virtue of the gas phaseextraction step means that the invention enables a far more rapid,economical, and accurate method for analyzing fluid samples using massspectrometry or other ionic detection devices.

Acoustic ejection, as noted above, enables rapid sample processing aswell as generation of nanoliter-sized droplets of predetermined andconsistent size; see U.S. Pat. No. 6,416,164 to Stearns et al., citedand incorporated by reference earlier herein. The aforementioned patentdescribes how the size of acoustically ejected droplets from a fluidsurface can be carefully controlled by varying the acoustic power, theacoustic frequency, the toneburst duration, and/or the F-number of thefocusing lens. An additional advantage of using acoustic ejection inconjunction with the present invention is that droplets can be ejectedfrom a very small sample size, on the order of 5 μl or less. This isparticularly advantageous when sample availability is limited and asmall fluid sample must be analyzed out of necessity. In terms ofprocessing capability, U.S. Pat. No. 6,938,995 to Mutz et al. explainsthat acoustic ejection technology, used in conjunction with acousticassessment of fluid samples in a plurality of reservoirs, can achieveanalysis of over 5, 10, or even 25 reservoirs per second, translating towell in excess of 50,000 fluid samples per day.

In one embodiment, then, the improved method of the invention makes useof an acoustic ejector as a fluid sample droplet generation device, thedevice including at least one reservoir to contain the fluid sample, anacoustic ejector, and a means for positioning the acoustic ejector inacoustic coupling relationship with the reservoir. Typically, a singleejector is used that is composed of an acoustic radiation generator anda focusing means for focusing the acoustic radiation generated by theacoustic radiation generator. However, a plurality of ejectors may beadvantageously used as well. Likewise, although a single reservoir maybe used, the device typically includes a plurality of reservoirs.

Examples of acoustic ejection devices useful in conjunction with thepresent invention are described in detail in U.S. Pat. No. 6,802,593 toEllson et al., U.S. Pat. No. 7,270,986 to Mutz et al., U.S. Pat. No.7,439,048 to Mutz et al., and U.S. Pat. No. 6,603,118 to Ellson et al.,incorporated by reference herein. As described therein, an acousticejection device may be constructed to include multiple reservoirs as anintegrated or permanently attached component of the device. However, toprovide modularity and interchangeability of components, it is preferredthat device be constructed with removable reservoirs. Generally, thereservoirs are arranged in a pattern or an array to provide eachreservoir with individual systematic addressability. In addition, whileeach of the reservoirs may be provided as a discrete or stand-aloneitem, in circumstances that require a large number of reservoirs, it ispreferred that the reservoirs be attached to each other or representintegrated portions of a single reservoir unit. For example, thereservoirs may represent individual wells in a well plate. Many wellplates suitable for use with the device are commercially available andmay contain, for example, 96, 384, 1536, or 3456 wells per well plate,having a full skirt, half skirt, or no skirt. Well plates or microtiterplates have become commonly used laboratory items. The Society forLaboratory Automation and Screening (SLAS) has established and maintainsstandards for microtiter plates in conjunction with the AmericanNational Standards Institute, including the footprint and dimensionstandards ANSI/SLAS 1-2004. The wells of such well plates typically formrectilinear arrays.

However, the availability of such commercially available well platesdoes not preclude the manufacture and use of custom-made well plates inother geometrical configurations containing at least about 10,000 wells,or as many as 100,000 to 500,000 wells, or more. Furthermore, thematerial used in the construction of reservoirs must be compatible withthe fluid samples contained therein. Thus, if it is intended that thereservoirs or wells contain an organic solvent such as acetonitrile,polymers that dissolve or swell in acetonitrile would be unsuitable foruse in forming the reservoirs or well plates. Similarly, reservoirs orwells intended to contain DMSO must be compatible with DMSO. Forwater-based fluids, a number of materials are suitable for theconstruction of reservoirs and include, but are not limited to, ceramicssuch as silicon oxide and aluminum oxide, metals such as stainless steeland platinum, and polymers such as polyester, polypropylene, cyclicolefin copolymers (e.g., those available commercially as Zeonex® fromNippon Zeon and Topas® from Ticona), polystyrene, andpolytetrafluoroethylene. For fluids that are photosensitive, thereservoirs may be constructed from an optically opaque material that hassufficient acoustic transparency for substantially unimpairedfunctioning of the device.

In addition, to reduce the amount of movement and time needed to alignthe acoustic radiation generator with each reservoir or reservoir wellduring operation, it is preferable that the center of each reservoir belocated not more than about 1 centimeter, more preferably not more thanabout 1.5 millimeters, still more preferably not more than about 1millimeter and optimally not more than about 0.5 millimeter, from aneighboring reservoir center. These dimensions tend to limit the size ofthe reservoirs to a maximum volume. The reservoirs are constructed tocontain typically no more than about 1 mL, preferably no more than about1 μL, and optimally no more than about 1 nL, of fluid. To facilitatehandling of multiple reservoirs, it is also preferred that thereservoirs be substantially acoustically indistinguishable.

A vibrational element or transducer is used to generate acousticradiation. In some instances, the acoustic radiation generator iscomprised of a single transducer. In addition, the transducer may use apiezoelectric element to convert electrical energy into mechanicalenergy associated with acoustic radiation. Alternatively, multipleelement acoustic radiation generators such as transducer assemblies maybe used. For example, linear acoustic arrays, curvilinear acousticarrays or phased acoustic arrays may be advantageously used to generateacoustic radiation that is transmitted simultaneous to a plurality ofreservoirs.

An added element in the form of a gas phase extraction device thermallyvolatilizes droplets ejected using an acoustic radiation generator asjust described, and extracts unwanted species from the droplets prior toanalysis using, e.g., a mass spectrometer. Replacement of conventionallyused components with more volatile substitutes (e.g., buffer salts) orsubstitutes that undergo a chemical reaction to provide volatilespecies, facilitates ready gas phase extraction of these components,thus eliminating or at least substantially reducing ion suppression orother types of interference seen with the conventional compounds.

As noted earlier herein, the method of the invention employs asubstitute component for the original non-analyte component that servesthe same purpose as the original component but is either (1) morevolatile than the original component and/or the analyte or (2) undergoesa chemical reaction upon volatilization to yield a reaction product thatis more volatile than the original component and/or the analyte.Embodiment (1) is discussed above. Now turning to embodiment (2), thesubstitute component is in this case not necessarily more volatile thanthe original component and/or the analyte, but is rather selected toundergo a chemical reaction that yields at least one volatile reactionproduct. The substitute component may be a zwitterionic compound thatundergoes an intramolecular conversion, as will be discussed in detailbelow, or the substitute component may be a nonzwitterionic compoundthat can be chemically cleaved to yield a volatile reaction product.Alternatively, the substitute component may be a compound that undergoesa thermally induced reaction to give rise to at least one volatilereaction product. The compound may also be selected to undergo aphotocatalytic reaction that results in at least one volatile reactionproduct, where, it is to be understood, the term “volatile reactionproduct” refers to a reaction product that is more volatile than theoriginal component and/or the analyte.

Zwitterionic Compound as the Substitute Component:

When a zwitterionic compound is used as the substitute component, e.g.,as a buffer salt in a fluid sample requiring a buffer system, thezwitterionic compound is selected so as to undergo a chemical reactionat elevated temperature and/or under reduced pressure during thevolatilization process, to provide at least one reaction product that ismore volatile than the original non-analyte component and/or theanalyte. The zwitterionic compounds described infra are useful in othermethods as well, e.g., in any method that involves ionization andvolatilization of a buffered fluid sample followed by detection of theanalyte in the ionized and volatilized sample.

By “zwitterion” or a “zwitterionic” compound as those terms are usedherein is meant a compound that contains a pair of ionizable groups, onethat ionizes to form a positively charged species and the other thationizes to form a negatively charged species. The former is typically anitrogen atom-containing group such as a substituted or unsubstitutedamine or a diazo substituent, and the latter is generally although notnecessarily a carboxyl (COOH) substituent. It will be appreciated thatat higher pH values only the nitrogen atom-containing group will bear acharge, such that the compound is cationic, while at lower pH valuesonly the carboxyl-containing group will be ionized, such that thecompound is anionic. At intermediate pH values, generally in the rangeof about 5 to about 8, both entities bear a charge and it is in thisform that the reactions described below proceed in an optimal manner.

In one embodiment, the zwitterionic compound is a partially unsaturatedcompound, i.e., a compound that contains at least one unsaturated bond,such as a double bond, and is “pre-aromatic” in the sense that theintramolecular decomposition results in the generation of a volatilearomatic compound. For instance, the partially unsaturated, pre-aromaticzwitterionic compound may comprise a partially unsaturated, pre-aromaticcore to which a carboxylic acid group (—COOH) and a nitrogen-containingsubstituent are covalently bound, wherein the nitrogen-containingsubstituent may be selected from amino, primary amino, secondary amino,tertiary amino, and diazo. In this example, the carboxylic acid groupand the nitrogen-containing substituent are positioned with respect toeach other so that the intramolecular decomposition yields a volatilearomatic compound (i.e., an aromatic compound that is more volatile thanthe original component and/or the analyte), releases carbon dioxide, andgenerates a nitrogen-containing compound such as ammonia, a substitutedor unsubstituted amine (generally gaseous), or nitrogen gas. Thepre-aromatic core is generally cyclic, although an acyclic core issuitable provided that the aforementioned intramolecular decompositionreaction results in a volatile compound, preferably a volatile aromaticcompound, in addition to release of carbon dioxide and anitrogen-containing compound. This pre-aromatic zwitterionic compoundcontaining pre-aromatic core Q and the nitrogen-containing substituentN* may be represented by structure (1)

When the core Q is cyclic, transformation from the pre-aromatic compoundcan occur according to the following scheme (I) when the carboxylategroup and the N* substituent are bound to adjacent carbon atoms in aring:

In Scheme (I), Ar represents the volatile aromatic reaction product, andeach N** reaction product corresponds to each N* substituent. Thisreaction may be illustrated using a more specific example in which,solely for purposes of illustration, the core Q comprises acyclohexa-1,3-diene ring, such that, as shown in Scheme (II), thevolatile aromatic reaction product is benzene:

The N* substituent, in cationic form, may be, for instance,—(NH₃)⁺—(NHR¹)⁺, (NR²R³)⁺, (NR⁴R⁵R⁶)⁺, or diazo, such that the N** willbe, respectively, ammonia (NH₃), NH₂R¹, NHR²R³, NR⁴R⁵R⁶, or nitrogen gas(N₂). R¹ through R⁶ are non-hydrogen substituents such as lower alkyl,i.e., a C₁-C₆ alkyl group, preferably a C₁-C₃ alkyl group, that may ormay not be substituted with substituents that will not interfere in thedetection process or interact with any of the components of the samplein a deleterious manner. The decomposition reaction thus results in twoproducts that will not be detected, i.e., the volatile aromatic compoundand carbon dioxide, and to the nitrogenous species N** which, isunlikely to cause ion suppression.

In Scheme (II), the cyclohexa-1,3-diene reactant may or may not besubstituted with one or more ring substituents that do not interferewith the chemical reaction shown or with any other compounds that arepresent, and are generally selected from the same group of substituentsas R¹ through R⁶, and the carboxylate and cationic nitrogen-containingsubstituent that are shown as ortho to each other may be in either cisor trans relationship (compound (2))

but are preferably in the trans configuration (compound (3)):

The optionally present substituents are illustrated in structure (4)

in which, as shown, there are i substituents where i is in the range ofzero to 6 inclusive, wherein the substituents, represented as R, may bethe same or different. In one particular case, wherein N* is diazo, itis preferred that the carboxylate and diazo substituent are in the transposition with a substituent attached to the same carbon as the diazogroup, where that substituent, shown as R⁷ in compound (5), is C₁-C₆alkyl, preferably C₁-C₃ alkyl (omission of R⁷ results in a compound thatis too unstable for the present purpose):

In a related embodiment, the zwitterionic compound is an aromaticcompound substituted with a carboxylate group and a diazo group, such asortho-diazo benzoic acid, in which case the intramolecular reactionresults in the N** reaction product benzyne (Scheme (III):

Benzyne is a very reactive compound that will serve as an intermediateto further reaction. In this case, a second reactant, e.g., water, or adiene, is introduced into the sample to react with benzyne. Water willresult in reaction to give phenol, while proper selection of the diene,as will be understood by a skilled practitioner, will yield a volatilereaction product. As one example, furan may serve as the diene, in whichcase reaction with benzyne gives the volatile product1,2,3,4-tetrahydro-1,4-epoxynaphthalene. As another example, anthracenemay serve as the diene, resulting in the volatile reaction producttriptycene.

The zwitterionic compound may be cyclic or acyclic, and, if cyclic, itmay be monocyclic, bicyclic, or polycyclic, and may contain aromaticrings as well as a molecular segment that is only partially unsaturated.When the chemical reaction of the zwitterionic compound results in avolatile aromatic compound as a reaction product, this is generally,although not necessarily, by addition of a double bond to a ringstructure to generate 4n+2 aromaticity. Examples include addition of adouble bond to a substituted or unsubstituted cyclohexyl-1,3-dienyl ringto generate a benzene ring, or addition of a double bond to asubstituted or unsubstituted dihydrofuran ring to generate furan, anaromatic. Thus, the zwitterionic compound may, in one instance, comprisea cyclohexyl-1,3-dienyl core that is substituted with the —COOH and —N*moieties at adjacent carbon atoms, such that the compound comprises5-carboxyl-6-N*-cyclohexa-1,3-diene, e.g.,5-carboxyl-6-amino-cyclohexa-1,3-diene, which may be further substitutedas indicated above, and which converts to a substituted or unsubstitutedbenzene ring following reaction. A further zwitterionic compound maycomprise a cyclohexa-1,4-diene core substituted with —COOH and —N* inthe para configuration, such that the compound is or contains3-carboxyl-6-N*-cyclohexa-1,4-diene, either unsubstituted or substitutedas above, which converts via the decomposition reaction to a substitutedor unsubstituted benzene ring. Another zwitterionic compound maycomprise, for instance, dihydrofuran substituted at the 2-position and3-position with the —COOH and —N* moieties, such that the compoundcomprises 2-carboxyl-3-N*-2,3-dihydrofuran or2-N*-3-carboxyl-2,3-dihydrofuran, either unsubstituted or unsubstitutedas described earlier herein, which, upon reaction, converts tosubstituted or unsubstituted furan, an aromatic molecule.

Specific examples of these zwitterionic compounds include, withoutlimitation, the following (for simplicity, the compounds are shown inuncharged form; it will be understood, however, that at intermediate pHvalues each compound will contain both an anionic species and a cationicspecies, i.e., a carboxylate group —COO and a positively chargednitrogen atom):

6-Amino-3,4-dimethylcyclohexa-2,4-diene-1-carboxylic Acid

1,6-Diamino-2,4-diene-1-carboxylic Acid

6-Amino-1,4-dimethylcyclohexa-2,4-diene-1-carboxylic Acid

6-Aminocyclohexa-2,4-diene-1-carboxylic Acid

6-(Dimethylamino)cyclohexa-2,4-diene-1-carboxylic Acid

6-(Ethylamino)cyclohexa-2,4-diene-1-carboxylic Acid

3-Amino-2,3-dihydrofuran-2-carboxylic Acid

4-Aminocyclohexa-2,5-dienecarboxylic Acid

10-Amino-9,10-dihydroanthracene-9-carboxylic Acid

4-Amino-1,4-dihydronaphthalene-1-carboxylic Acid

2-Amino-1,2-dihydronaphthalene-1-carboxylic Acid

2-Amino-1,2-dihydronaphthalene-1-carboxylic Acid

4-(Ethylamino)-1,4-dihydronaphthalene-1-carboxylic Acid

These zwitterionic compounds may be obtained commercially or synthesizedusing methods known in the art and described in the pertinentliterature. Carboxylate-containing zwitterions can be synthesized usingany of a variety of techniques for combining a carboxyl-containingcompound with an amine or other nitrogenous compound. Zwitterionicsulfonic acid-containing compounds such as zwitterionic detergents andbuffers can generally be synthesized from substituted or unsubstituted1,2-oxathiolane-2,2-dioxide and a substituted or unsubstituted amine,according to Scheme (IV):

Other Chemically Cleavable Substitute Components:

Alternatively, a chemically cleavable nonzwitterionic compound can beselected to serve as the substitute component, wherein the chemicallycleavable compound contains at least one linkage that can be cleavedwith an acid, base, or other reagent. Representative linkageshydrolytically cleavable in the presence of acids or bases include thefollowing: carboxylate ester (—(CO)—O—); enol ether (—CH═CH—O—); acetal(—O—CR₂—O—); hemiacetal (—CH(OH)—O—); anhydride (—(CO)—O—(CO)—);carbonate (—O—(CO)—O—); amide ((—(CO)—NH—); N-substituted amide(—(CO)—NR—); urethane (—O—(CO)—NH—); N-substituted urethane(—O—(CO)—NR—); imido (—CH═N—); N,N-disubstituted hydrazo (—NR—NR—);thioester (—(CO)—S—); phosphonic ester (—P(O)(OR)—O—); sulfonic ester(—SO₂—OR—); ortho ester (—C(OR)₂—O—); and betaine ester (R—O—(CO)—N(W)₄⁺X⁻ where the R′ may be the same or different non-hydrogen substituentsand X⁻ is the associated counterion). Other chemically cleavablelinkages include, without limitation: the hydroxylamine-cleavablelinkage —(CO)—O—CH₂—CH₂—O—(CO)—; the thiol-cleavable linkage —S—S, alsocleavable upon treatment with trisubstituted phosphines such astriphenylphoephine; periodate cleavable cis-diols —CH(OH)—CH)OH—; andthe fluoride-cleavable linkage—(O)—NH—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—(NH)—(CO)—. Surfactants containingchemically cleavable linkages have been described, along with synthesisthereof. these include ProteaseMAX (V2071) (Promega); RapiGest SF(Waters); PPS Silent Surfactant (Agilent); MaSDeS (see Chang et al.(January 2015) J. Proteome Res. 14(3)); Invitrosol (Life Technologies,Inc.); Progenta AALS I (sodium 2,2-dihexoxypropyl sulfate, from ProteaBiosciences); Progenta AALS II (sodium 2,2-diheptoxypropyl sulfate, alsofrom Protea Biosciences); Progenta CALS I (2,2-dihexoxypropyl ammoniumbromide, also from Protea Biosciences); and Progenta CALS II(2,2-diheptoxypropyl ammonium bromide, also from Protea Biosciences).These surfactants and analogs thereof can be used advantageously in thepresent compositions, as can buffers and other compounds containingthese and other chemically cleavable linkages.

Thermally Cleavable Substitute Components:

Substitute components can also contain linkages that are cleavable withheat, such that they serve the same purpose as the original component inthe fluid sample but cleave into smaller compounds upon volatilizationof the sample, where those smaller compounds are either volatile orunlikely to cause ion suppression. Thermally cleavable linkages includeester linkages, carbamate linkages, carbonate linkages, urethane-typelinkages (—O—(CO)—NH—) and N-substituted urethane linkages (—O—(CO)—NR—)in which the nitrogen atom of the linkage is substituted with anon-hydrogen substituent such as lower alkyl. Other thermally cleavablelinkages include furan-maleimide Diels-Alder adducts (see Szalal et al.(2007) Macromolecules 40(4): 818-823), oxirane and thiirane-basedlinkages, and ester-substituted sulfones that thermally decompose to anester and gaseous SO₂ according to the following scheme

wherein R is a nonhydrogen substituent, generally an alkyl group (e.g.,piperylene, as described by Eckert and Liotta, “Designing SmartSurfactants” printed fromwww.chbe.gatech.edu/eckert/pdf/-surfactant.pdf, Internet site accessedon Oct. 19, 2015).

Photolytically Cleavable Substitute Components:

Incorporation of one or more photolytically cleavable sites into thesubstitute component allows for irradiation-induced cleavage prior tointroduction of the sample into the mass spectrometer or otheranalytical device. The sample fluid or fluids can be irradiated in thegas phase, i.e., after fluid droplet ejection but prior to entry intothe mass spectrometer, or they may be irradiated in the liquid phase,e.g., in a well plate or other container or group of containers.Irradiation in the gas phase enables real-time conversion to thecleavage products, while irradiation in the liquid phase, where samplefluid is present in a multiplicity of containers or wells, does not.

Photolytically cleavable sites can be readily incorporated into thesubstitute component, e.g., the buffer, surfactant, or the like, usingsynthetic organic techniques known to those of ordinary skill in the artand/or described in the pertinent texts and literature. One type ofphotolytically cleavable linkage is composed of an ortho-nitrobenzylgroup as in the following representative structure

where R* is generally a nitrogen atom or oxygen atom bound to the restof the molecule. Such structures thus include

and

as well as the N-substituted analog

in which R, again, is a non-hydrogen substituent such as lower alkyl,and the

symbol represents attachment to the remainder of the molecule. Anotherphotolytically cleavable linkage includes, by way of example, thecinnamic acid-type linkage

described, for example, by Sakai et al. (15 Jun. 2012) J. Colloid andInterface Sci. 376(1):160-164, with respect to the photocleavablesurfactant

Other photolytically cleavable linkages include benzyl ethers (whichcleave to form alcohols), carbamate linkages (which cleave to formamines), 1,3-dithiane linkages (which cleave to form carbonyl groups);ortho-nitroanilide linkages (which cleave to form carboxyl groups),benzoin-type linkages (which cleave to form phosphate groups), and thelike. See, e.g., Pelliccioli et al. (2002) Photochem. Photobiol. Sci.1:441-458, and Greene et al., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons (New York, N.Y.: 1999).

Those of ordinary skill in the art will be able to use known methods oforganic synthesis and/or methods described in the literature tosynthesize suitable zwitterionic and/or cleavable compounds that can beused as the substitute component herein. Additionally, known buffers(e.g., the Good's buffers; see Good et al. (1966) Biochemistry5(2):467-477), surfactants, and the like may be modified to incorporatesuch cleavable linkers.

One representative buffer of interest is the acetal-containing compound(25)

which decomposes photolytically and/or in the presence of acid asfollows to give 2-aminoethanol and 2-formylbenzoic acid. Another bufferof interest is compound (26)

which can be photolytically cleaved to give methamine (CH₃—NH₂) and4-formyl-3-nitrosobenzoic acid.

Compounds having the general structure

are suitable substitute components herein, particularly as anacid-cleavable buffer. In (27), L¹ and L² are C₁-C₆ hydrocarbyllinkages, generally C₂-C₄ hydrocarbyl, R⁸, R⁹, R¹⁰, and R¹¹ areindependently selected from H and C₁-C₁₆ hydrocarbyl (e.g., alkyl,cycloalkyl, alkenyl, etc., particularly lower alkyl), R¹² is loweralkyl, and R¹³ is either —COOH or —CH₂OSO₃H. When R¹³ is —COOH, thecompound may be represented as (28), while when R¹³ is —CH₂OSO₃H, thecompound may be represented as (29):

In a preferred embodiment, R⁸, R⁹, R¹⁰, and R¹¹ are H, L¹ and L² are—CH₂CH₂—, and R¹² is methyl, such that generic compounds (28) and (29)have the structures (30) and (31), respectively:

Both compounds can be synthesized from methyl pyruvate. Compound (30)has a pI of approximately 9.44 and is optimally employed as a buffer ata pH in the range of approximately 7.5 to 10.5. Compound (31) has a pIof approximately 9.44 and is optimally employed as a buffer at a pH inthe range of approximately 8.0 to 11.0. FIG. 4 schematically illustratesthe synthesis of compound (30), while FIG. 5 schematically illustratesthe synthesis of compound (31). Implementation of the individualreaction steps shown therein will be within the purview of those skilledin the art and/or will become apparent upon reference to an analogousreaction in the texts or literature.

It is to be understood that while the invention has been described inconjunction with a number of specific embodiments, the foregoingdescription as well as the examples that follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications will be apparent to those skilled in theart. All patents, patent applications, and publications mentioned hereare hereby incorporated by reference in their entireties.

Example 1

In this example, the impact of buffer volatility on the massspectrometric determination of caffeine was evaluated using ammoniumacetate as a volatile buffer salt and sodium chloride as a nonvolatilebuffer salt. Fluid samples were prepared with varying concentrations ofeither ammonium acetate or sodium chloride, at 1 mM, 2 mM, 5 mM, 10 mM,25 mM, 50 mM, 100 mM, 250 mM, and 500 mM. Sample droplets were generatedusing a modified Echo® 555 liquid handler (Labcyte Inc., Sunnyvale,Calif.). The instrument was modified to move the transducer assembly tothe exterior of the instrument underneath a nozzle to allow the dropletstream to be exposed to 195° C. heat, thus enabling gas phase extractionof volatile salt, and pulled into a beta version of the SQ Detector IImass spectrometer (Waters Micromass).

FIG. 1 is a plot of normalized signal at m/z=195 versus ammonium acetate(♦) or sodium chloride (▪) concentration (mM). As may be seen, theconcentration of caffeine detected decreases rapidly at even low sodiumchloride concentrations, a phenomenon not seen with ammonium acetate.This result indicates that the more volatile buffer salt allows foranalyte detection, even at higher buffer salt concentrations, while theless volatile buffer salt does not.

Experimental for Examples 2 and 3

All samples were analysed on a Waters SQ Detector 2 mass spectrometerfitted with a standard electrospray source running a source blocktemperature of 80° C., a desolvation temperature of 250° C. and a gasflow rate of 400 liters/hr. The electrospray probe voltage was set to3500 V and the sample cone to 25 V. All samples were introduced using aHarvard 22 syringe pump fitted with a Hamilton 250 μl syringe operatingat 6 μl/min. All reagents were purchased from Sigma Aldrich as drypowders except HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid, a zwitterionic buffer), which was supplied as a 1M solution. 1Mstock solutions were prepared for each of the salts using HPLC gradewater and a caffeine stock solution of 2 mg/ml, again in HPLC gradewater. Serial dilutions were performed on each of the salt stocksolutions to generate 1 mM, 5 mM, 25 mM and 50 mM working solutions. To1 ml of each of these working solutions 50 pl of the caffeine stocksolution was added to produce a 10 μg/ml caffeine solution for analysis.For each sample a syringe infusion was performed and the absolute ionsignal heights for the M+H and M+Na peaks of caffeine (at 195 Da and 217Da) were summed and plotted. Between each sample, the syringe and probewere flushed with water to remove any salt residues.

Example 2

This example describes an additional evaluation of the impact of buffervolatility on the mass spectrometric determination of caffeine, withmagnesium acetate used as a volatile buffer salt and magnesium chlorideas a nonvolatile buffer salt. The signal intensity of caffeine and itssodium adduct was evaluated in four concentrations of six salt systems.The data, plotted in FIG. 2, shows that standard ESI system sensitivityis heavily impacted by non-volatile salts and less impacted by avolatile salt, at similar concentrations. That is, the uppermost datapoints in the plot correspond to the signal obtained with fluid samplescontaining caffeine and ammonium acetate as a buffer salt. Theexperiment may be repeated to carry out mass spectrometricdeterminations of other analytes with substantially the same results.

Example 3

In this example, a kinase assay was conducted in which magnesium isrequired by the enzyme used, and the assay measures the increase inconcentration of a phosphorylated peptide substrate with time. One setof samples was formulated with magnesium chloride, a relativelynonvolatile compound, and a second set of samples was formulated withmagnesium acetate, a more volatile compound. The assay results usingeach type of magnesium salt are illustrated in FIG. 3, in which time, inminutes, is represented on the X-axis, and luminescence is representedon the Y-axis. The graph comparing results obtained for the assayemploying magnesium chloride (♦) with those obtained for the assayemploying magnesium acetate (▪) show that the assay was not hindered orbiased by the source of magnesium ions.

In many assay systems, certain non-volatile and/or highlyelectronegative buffer components are used; however, these componentsare not unique creating the biological or chemical outcome of thedesired assay. The switch does not show any significant change inresults for the range of up to 60 as this is within the noise of themeasurement. From the previous examples for measurement of analyte withthe absence of the non-volatile analyte, this assay system would provideimproved results with gas phase extraction prior to MS loading due toreduce ion suppression in the analyte.

1. An improved method for determining the concentration of an analyte ina fluid sample that additionally comprises a necessary non-analyteoriginal component, the method comprising volatilizing and ionizing thesample, and introducing the ionized, volatilized sample into an ionicanalyte detection device that provides a signal proportional inintensity to the quantity of ionized analyte detected, wherein theimprovement comprises: acoustically generating nanoliter-sized dropletsof the fluid sample prior to volatilization and ionization, such thatthe fluid sample is introduced into the ionic analyte detection devicein the form of nanoliter-sized droplets; and substituting for thenecessary non-analyte original component a substitute component that:(a) functions as the original component in the fluid sample; (b) (i) ismore volatile than the necessary original component or (ii) uponvolatilizing the fluid sample, undergoes a reaction to yield at leastone reaction product that is more volatile than the necessary originalcomponent; and (c) results in an increase in the intensity of the signaland/or a greater signal-to-noise ratio than either the signal intensityor signal to noise ratio obtained using the original component. 2.(canceled)
 3. The method of claim 1, wherein the nanoliter-sizeddroplets of fluid sample have a mean droplet size of less than aboutapproximately 5 nl.
 4. The method of claim 3, wherein thenanoliter-sized droplets of fluid sample have a mean droplet size ofless than about approximately 2.5 nl.
 5. The method of claim 4, whereinthe nanoliter-sized droplets of fluid sample have a mean droplet size ofless than about approximately 50 pl.
 6. The method of claim 5, whereinthe nanoliter-sized droplets of fluid sample have a mean droplet size ofless than about approximately 1 pl.
 7. The method of claim 1, whereinthe improvement further includes using focused acoustic ejection togenerate the nanoliter-sized droplets of the fluid sample.
 8. The methodof claim 7, wherein the acoustic ejection is carried out using anacoustic ejector that directs focused acoustic energy into a reservoircontaining the fluid sample in a manner that results in the rapidejection of consistently sized fluid droplets from the surface of thefluid sample.
 9. The method of claim 1, wherein the ionic analytedetection device comprises a mass spectrometer.
 10. The method of claim9, wherein the ionizing comprises chemical ionization, field desorptionionization, electrospray ionization, atmospheric pressure chemicalionization, matrix-assisted laser desorption ionization, or inductivelycoupled plasma ionization.
 11. The method of claim 1, wherein theanalyte comprises a drug, a metabolite, an inhibitor, a ligand, areceptor, a catalyst, a synthetic polymer, or an allosteric effector.12. The method of claim 1, wherein the analyte is a biomolecule.
 13. Themethod of claim 12, wherein the biomolecule comprises a nucleotideanalyte, a peptidic analyte, or a saccharidic analyte.
 14. The method ofclaim 1, wherein the necessary non-analyte original component comprisesan original salt and the substitute component comprises a substitutesalt.
 15. The method of claim 14, wherein the original salt and thesubstitute salt function as buffer salts for the fluid sample, such thatvolatilization of the fluid sample results in gas phase extraction ofthe substitute buffer salt.
 16. The method of claim 15, wherein thesubstitute salt comprises singly charged ions formed from weak acids orweak bases.
 17. The method of claim 16, wherein the substitute saltcomprises ammonium bicarbonate, ammonium formate, ammonium acetate,pyridinium acetate, pyridinium formate, ethylmorpholinium acetate,trimethylamino acetate, or trimethylamino formate.
 18. The method ofclaim 17, wherein the substitute salt comprises ammonium bicarbonate,ammonium formate, or ammonium acetate. 19-40. (canceled)
 41. The methodof claim 1, where the increase in analyte signal intensity and/orsignal-to-noise ratio is at least 10%.
 42. The improved method of claim41, where the increase in analyte signal intensity and/orsignal-to-noise ratio is at least 25%.
 43. The method of claim 1,wherein the droplets introduced into the ionic analyte detection devicecomprise the analyte and the substitute component.
 44. An improvedmethod for determining the concentration of an analyte in each of aplurality of fluid samples that additionally comprises a necessarynon-analyte original component, the method comprising volatilizing andionizing the samples and introducing each ionized, volatilized sampleinto an ionic analyte detection device that provides a signalproportional in intensity to the quantity of ionized analyte detected,wherein the improvement comprises substituting for the necessarynon-analyte original component a substitute component that functions asthe original component in the fluid sample, is more volatile than thenecessary original component, and results in an increase in theintensity of the signal and/or a greater signal-to-noise ratio thaneither the signal intensity or signal to noise ratio obtained using theoriginal component, and additionally comprises (a) providing the fluidsamples in each of a plurality of fluid reservoirs; (b) acousticallycoupling an acoustic droplet ejector to a first of the fluid reservoirs;(c) activating the ejector to generate focused acoustic radiation towardthe first reservoir and into the fluid sample therein, in a mannereffective to eject nanoliter-sized droplets of the fluid sample into theionic analyte detection device; (d) positioning another of the fluidreservoirs and the acoustic droplet ejector in acoustic couplingrelationship; (e) repeating step (c); and (f) repeating steps (d) and(e) with additional fluid reservoirs in the plurality of fluidreservoirs at a rate of greater than 5 reservoirs per second.
 45. Themethod of claim 44, wherein the droplets comprise both the analyte andthe substitute component.
 46. The method of claim 44, wherein thenanoliter-sized droplets of fluid sample have a mean droplet size ofless than about approximately 5 nl.
 47. The method of claim 46, whereinthe nanoliter-sized droplets of fluid sample have a mean droplet size ofless than about approximately 2.5 nl.
 48. The method of claim 47,wherein the nanoliter-sized droplets of fluid sample have a mean dropletsize of less than about approximately 50 pl.
 49. The method of claim 48,wherein the nanoliter-sized droplets of fluid sample have a mean dropletsize of less than about approximately 1 pl.
 50. The method of claim 44,wherein the rate is greater than 10 reservoirs per second.
 51. Themethod of claim 50, wherein the rate is greater than 25 reservoirs persecond.
 52. The method of claim 44, wherein the ionic analyte detectiondevice comprises a mass spectrometer.
 53. The method of claim 52,wherein the ionizing comprises chemical ionization, field desorptionionization, electrospray ionization, atmospheric pressure chemicalionization, matrix-assisted laser desorption ionization, or inductivelycoupled plasma ionization.
 54. The method of claim 44, wherein theanalyte comprises a drug, a metabolite, an inhibitor, a ligand, areceptor, a catalyst, a synthetic polymer, or an allosteric effector.55. The method of claim 44, wherein the analyte is a biomolecule. 56.The method of claim 44, wherein the biomolecule comprises a nucleotideanalyte, a peptidic analyte, or a saccharidic analyte.
 57. The method ofclaim 44, wherein the necessary non-analyte original component comprisesan original salt and the substitute component comprises a substitutesalt.
 58. The method of claim 57, wherein the original salt and thesubstitute salt function as buffer salts for the fluid sample, such thatvolatilization of the fluid sample results in gas phase extraction ofthe substitute buffer salt.
 59. The method of claim 58, wherein thesubstitute salt comprises singly charged ions formed from weak acids orweak bases.
 60. The method of claim 59, wherein the substitute saltcomprises ammonium bicarbonate, ammonium formate, ammonium acetate,pyridinium acetate, pyridinium formate, ethylmorpholinium acetate,trimethylamino acetate, or trimethylamino formate.
 61. The method ofclaim 60, wherein the substitute salt comprises ammonium bicarbonate,ammonium formate, or ammonium acetate.
 62. The method of claim 44,wherein the fluid reservoirs are arranged in an array.
 63. The method ofclaim 62, wherein the fluid reservoirs are contained within a substratecomprising an integrated multiple reservoir unit.
 64. The method ofclaim 63, wherein the integrated multiple reservoir unit is a microwellplate and the fluid reservoirs are wells therein.
 65. The method ofclaim 62, wherein the fluid reservoirs are tubes in a tube rack.
 66. Animproved method for determining the concentration of an analyte in eachof a plurality of fluid samples that additionally comprises a necessarynon-analyte original component, the method comprising volatilizing andionizing the samples and introducing each ionized, volatilized sampleinto an ionic analyte detection device that provides a signalproportional in intensity to the quantity of ionized analyte detected,wherein the improvement comprises substituting for the necessarynon-analyte original component a substitute component that functions asthe original component in the fluid sample, is more volatile than thenecessary original component, and results in an increase in theintensity of the signal and/or a greater signal-to-noise ratio thaneither the signal intensity or signal to noise ratio obtained using theoriginal component, and additionally comprises (a) providing the fluidsamples in each of a plurality of fluid reservoirs; (b) acousticallycoupling an acoustic droplet ejector to a first of the fluid reservoirs;(c) activating the ejector to generate focused acoustic radiation towardthe first reservoir and into the fluid sample therein, in a mannereffective to eject nanoliter-sized droplets of the fluid sample into theionic analyte detection device, wherein the droplets have a mean dropletsize of less than approximately 50 pl; (d) positioning another of thefluid reservoirs and the acoustic droplet ejector in acoustic couplingrelationship; (e) repeating step (c); and (f) repeating steps (d) and(e) with additional fluid reservoirs in the plurality of fluidreservoirs at a rate of greater than 10 reservoirs per second.